Comparison of LIRADS 5 Image Guided Core Biopsy Derived From Formalin Fixed and Frozen Tissue Cores for Radiogenomics and Radioproteomics Analysis in Well, Moderate and Poorly Differentiated Hepatocellular Carcinoma

The aim of this pilot study is to evaluate and compare the quality of the genomics and proteomics data obtained from paired Formalin Fixed Paraffin Embedded (FFPE) and frozen (FF) tissue percutaneous core biopsies of Liver Imaging Reporting and Data System 5 (LIRADS 5) hepatocellular carcinoma (HCC) of varying histological grades. The preliminary data identified differentially expressed proteins and genes in poor, moderate and well differentiated HCC biopsies, with a greater efficacy in fresh frozen samples. The data offered valuable insights into the characteristics and suitability of samples for future studies.


Introduction
Hepatocellular Carcinoma (HCC) is the most common liver cancer and it's the leading cause of cancer-related deaths worldwide.HCC patients do not respond to most systemic therapies [1,2].
Current diagnostic tests are blood, imaging, and biopsy, with no useful predictive biomarkers.Very few proteomics biomarkers have been studied for liver HCC and their correlation to clinical behavior and response to therapy is limited [3][4][5].Targeted therapies may prolong survival in a minority of patients but are not personalized [6,7].Therefore, we aim to analyze the tissue biopsies of HCC patients of different grades, using proteomics and genomics analysis, and combine it with our imaging CT/MRI data (radiomics) to generate radioproteomics and radiogenomics data to better predict HCC subtypes and ultimately offer personalized medicine for HCC patients.
Most of percutaneous image guided clinical biopsies are stored as FFPE, which is the standard method used by pathologists to diagnose histologically HCC grades.However, comparison studies of other type of cancer tissues, suggested that using FF tissues have advantages over FFPE, such as: proteins/ DNA/ RNA are better preserved, the variability if FF tissues are lower than FFPE tissues which can affect the data quality, and FF samples can be stored for more than 2 years with no risk of DNA/protein degradation unlike FFPE samples [8,9].
Here we report the preliminary data that we obtained by comparing three paired FFPE and FF tissue biopsies, derived from 18 G percutaneous biopsy of LIRADS 5, three histological grades (well differentiated, moderate differentiated, and poorly differentiated) HCC, to determine the most optimal tissue type for our larger study.

RNA-seq library construction and sequencing
RNA-seq libraries of three paired FFPE and FF tissue biopsies of three histological grades were prepared with KAPA mRNA HyperPrep Kit with RiboErase (Roche).rRNA was depleted by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase.The first strand cDNA synthesized using random priming followed by second strand synthesis converting cDNA:RNA hybrid to double-stranded cDNA (dscDNA), and incorporating dUTP into the second cDNA strand.cDNA generation is followed by end repair to generate blunt ends, A-tailing, adaptor ligation and PCR amplification.
Sequencing was performed on Illumina NovaSeq6000 for a paired end 2x50 run.Data quality check was done on Illumina SAV.Demultiplexing was performed with Illumina software.The reads were mapped by STAR 2.7.9a [10] and read counts per gene were quantified using the human genome GRCh38.104.In Partek Flow [9], read counts were normalized by CPM +1.0E-4.Differential expression of genes was measured using the gene set enrichment (GSA) algorithm in Partek Flow, generating unfiltered as well as filtered datasets.Statistical filters for differential expression were set at fold-change > 2 and p < 0.01.

Deparaffinization of FFPE Tissues
FFPE tissue scrolls were placed in Eppendorf tubes, 1 mL of 100% xylene was added for 10 minutes to deparaffinized the tissue scrolls.Centrifuged 3 times, at 16,000 x g for 3 min, supernatant was discarded.Followed by 3 mL of 100% ethanol, for 3 min, pelleted at 16,000 x g for 3 minutes.This step was repeated an additional two times.Supernatant was discarded.

Protein digestion and TMT labelling
Fresh frozen and FFPE tissue homogenization was carried out using 12 mM sodium lauryl sarcosine, 0.5% sodium deoxycholate, and 50 mM triethyl ammonium bicarbonate TEAB, in ultrasonic cell disruptor for 20 seconds.Samples were then centrifuged at 16,000 × g for 5 min, supernatant was collected, heated at 95°C for 1 hour and sonicated for 5 minutes.The total protein concentration of the samples was determined using BCA Protein Assay Kit (Pierce, Thermo Fischer Scientific).The standard curve was generated using Bovine serum albumin.The samples were treated with tris (2-carboxyethyl) phosphine (10 μL, 55 mM in 50 mM TEAB, 30 min, 37 °C), followed by treatment with chloroacetamide (10 μL, 120 mM in 50 mM TEAB, 30 min, 25 °C in the dark).They were then diluted five-fold with aqueous 50 mM TEAB and incubated overnight with Sequencing Grade Modified Trypsin (1 μg in 10 μL of 50 mM TEAB; Promega, Cat # V511A, Madison, WI, USA), 1 μg of trypsin per sample used regardless of protein extraction yield.After digestion an equal amounts of peptides TMT were labelled and combined for MS analysis, modified protocol from Simonian, et al. [11].An equal volume of ethyl acetate/trifluoroacetic acid (TFA, 100/1, v/v) was then added, followed by avigorous mix (5 min) and centrifugation (13,000 × g, 5 min).The supernatants were discarded, and the lower phases were dried in a centrifugal vacuum concentrator.The samples were then desalted using a modified version of Rappsilber's, et al. protocol [12], in which the dried samples were reconstituted in acetonitrile/water/TFA (solvent A, 100 μL, 2/98/0.1,v/v/v) and then loaded onto a small portion of a C18-silica disk (3M, Maplewood, MN, USA) placed in a 200 μL pipette tip.Prior to sample loading, the C18 disk was prepared by sequential treatment with methanol (20 μL), acetonitrile/ water/TFA (solvent B, 20 μL, 80/20/0.1,v/v/v), and finally with solvent A (20 μL).After loading the sample, the disc was washed with solvent A (20 μL, eluent discarded) and eluted with solvent B (40 μL).The collected eluent was dried in a centrifugal vacuum concentrator.The samples were then chemically modified using a TMT11plex Isobaric Label Reagent Set (Thermo Fisher Scientific, Cat # A34808, Waltham, MA, USA) as per the manufacturer's protocol.The TMT-labeled peptides were dried and reconstituted in solvent A (50 μL), and an aliquot (2 μL) was taken for measurement of total peptide concentration (Pierce Quantitative Colorimetric Peptide, Thermo Fisher Scientific, Waltham, MA, USA).The samples were then pooled and desalted again using the modified Rappsilber's protocol.

Proteomics Data Analysis
Raw proteomic data were searched against a Uniprot database containing the complete reference human proteome (ID: UP000005640, Gene Count: 20597) using SEQUEST-HT (including dynamic modifications: oxidation (+15.995) on M, deamidation (+0.984) on N/Q, and carbamidomethyl (+57.021),phosphorylation (+79.966) on S/T/Y) in Proteome Discoverer (Version 2,4, Thermo Scientific), which provided measurements of relative abundance of the identified peptides.Decoy database searching was used to generate high confidence tryptic peptides (FDR < 1%).Tryptic peptides containing amino acid sequences unique to individual proteins were used to identify and provide relative quantification between different proteins in each sample.

Results
RNA-seq identified 12,791 genes with 594 overlapping differentially expressed genes.The gene quantification efficiency of sequencing data gathered from FF was markedly higher than from FFPE tissues, with average gene counts per mapped reads of (0.61) vs (0.35) respectively (Figure 1).However, the overlapping upregulated genes in FFPE were higher than FF in the three histological grades, likely, due to tissue heat response associated with FFPE sample preparation.On histological grades comparison analysis, the overlapping upregulated genes in the moderately differentiated tissue cores were slightly higher than well differentiated tissue and markedly higher than poorly differentiated tissue cores in both FF and FFPE biopsy samples (Figures 2 and 3).
Additionally, 30 more genes were upregulated in moderate vs well differentiated in both FF and FFPE tissue cores, but with a fold-change < 2.   Heat map of all protein identified in FF and FFPE of liver HCC from proteomics analysis.The white patches indicates that proteins were not detected

Table 1 .
The significantly upregulated genes > 2 fold, in moderate vs well differentiated tissue cores in both FF and FFPE, with greater fold change in FF, due to higher gene counts

Table 2 .
Some of the upregulated proteins >1 fold, in (poor vs moderate) and (poor vs well) differentiated of the frozen tissues FF